The present invention relates to a poly ICLC formulation with improved therapeutic efficacy.
Double-stranded RNAs (dsRNAs) are very potent biologic modifiers. They can exert a profound influence on cells at nanomolar concentrations. The modulating effects of dsRNA include a broad spectrum of actions at the molecular and cellular levels. At the molecular level, dsRNAs can elicit biological effects such as interferon synthesis, induction of protein kinase, induction of 2-5A polymerase. enhancement of histocompatibility antigen and inhibition of metabolism. And at the cellular level, dsRNA can elicit biological effects such as pyrogenicity, mitogenicity, macrophage activation, activation of cell-mediated immunity and induction of antiviral state. One promising potential of dsRNAs is its immunomodulating effect in antimicrobial and anticancer therapies. In particular, the double-stranded RNA poly ICLC, or PICLC for short, was found highly effective as an antiviral or antitumor agent.
Poly ICLC is a synthetic dsRNA consisting of polyriboinosinic and polyribocytidylic acid strands (poly I.poly C) stabilized with poly-L-lysine and carboxymethylcellulose. The resulting poly ICLC is thermodynamically more stable than poly I.poly C. Poly ICLC has been shown in clinical trials to be effective in the cancer treatment of gliomas (Salazar, A, M. and al., Neurosurgery 38:1096-1104). It has also been shown in a number of studies to be effective in the immunotherapy of viral infection including influenza (Wong, J. P. Antimicrob. Agents Chemother, 39:574-2576), rabies (Baer, G. M., J. Infect. Dis. 136:286-292). Rift Valley fever (Kende, M., J. Biol. Response Modifiers 4:503-511) and Venequelan equine encephamyelitis (Stephen, E. L., J. Infect. Dis. 136:267-272).
Although poly ICLC is a promising immunomodulator which has great potential in antimicrobial and anticancer therapies, it has been shown to produce serious side effects in humans, especially when the drug is administered in multiple high doses. Some of the reported side effects (Levine, A. S., Cancer Treat. Rep. 62:1907-1913) include fever, hypotension, leukopenia, myalgia, thrombocytopenia and poly arthalgia. The inherent toxicity problem must be overcome to render poly ICLC safer for use in humans. Furthermore, the therapeutic efficacy of poly ICLC is limited by its stability in vivo. As a ribonucleic acid, poly ICLC is susceptible to degradation in the body by serum RNAse. Although the extent of RNAse degradation of poly ICLC is much improved as compare to that of poly I.poly C, the protection is not complete and poly-L-lysine and carboxymethylcellulose themselves may be susceptible to enzymatic degradation and immunological clearance in vivo. Therefore, a need exists for an improved formulation of poly ICLC which has improved therapeutic efficacy and will be safer for use in humans.
It is an object of the present invention to provide a poly ICLC formulation having enhanced therapeutic efficacy while reducing it toxic effect in humans.
In accordance with one aspect of the present invention, there is provided an immunomodulating agent comprising poly ICLC encapsulated within liposomes. Preferably, the liposomes used are unilamellar or multilamellar and contain at least one cationic phospholipid such as stearylamine, 1,2-diacyl-3-trimethylammonium-propane (TAP) or 1,2-triacyl-3-dimethylammonium-propane (DAP). Most preferably, the liposomes are unilamellar or multilamellar liposomes prepared from the lipids phosphatidylcholine and stearylamine, and the steroid cholesterol at a molar ratio of approximately 9:1:1, respectively. The surface liposomes may be coated with polyethylene glycol to prolong the circulating half-life of the liposomes, and with antibody for targeting to specific sites in the body.
Neutrally charged liposomes can also be used for liposomal entrapment of poly ICLC. Such neutrally charged liposomes can be prepared by using, for example phosphatidylcholine and cholesterol.
In accordance with another aspect of the present invention there is provided a method for preparing liposomal poly ICLC comprising the step of freeze-drying a mixture of liposomes and poly ICLC. Conveniently, the method includes removing organic solvent from a mixture of phospholipids, rehydrating the resulting lipids mixture with an aqueous buffer containing poly ICLC, freeze-drying the resulting lipid-poly ICLC mixture, reconstituting the resulting dried mixture, and resuspending the resulting liposome pellets with a buffer solution to the desired drug concentration prior to use. Suitable buffer can be phosphate buffered saline, normal saline or deionized water. It is important for the preparation of buffer solution to use RNAse-free water so that enzymatic degradation of poly ICLC can be minimized.
Alternate methods of preparation of liposomes include detergent dialysis, extrusion, reverse-phase evaporation (REV) and sonication. The loading of poly ICLC into the liposomes can be achieved by passive trapping and by active process such as remote loading. The unentrapped poly ICLC can be removed by centrifugation, column separation or by dialysis.
The advantages of encapsulating poly ICLC in liposomes are that the toxicity of poly ICLC is decreased, and at the same time the therapeutic efficacy of poly ICLC is increased. Furthermore, liposomal poly ICLC protects the poly ICLC from RNAse degradation in the body, thereby enhancing the immunological and biological activities of poly ICLC.